Assembly factors for the membrane arm of human complex I Byron Andrews, Joe Carroll, Shujing Ding, Ian M. Fearnley, and John E. Walker1 Medical Research Council Mitochondrial Biology Unit, Cambridge CB2 0XY, United Kingdom Contributed by John E. Walker, October 14, 2013 (sent for review September 12, 2013) Mitochondrial respiratory complex I is a product of both the nuclear subunits in a fungal enzyme from Yarrowia lipolytica seem to be and mitochondrial genomes. The integration of seven subunits distributed similarly (12, 13). encoded in mitochondrial DNA into the inner membrane, their asso- The assembly of mitochondrial complex I involves building the ciation with 14 nuclear-encoded membrane subunits, the construc- 44 subunits emanating from two genomes into the two domains of tion of the extrinsic arm from 23 additional nuclear-encoded the complex. The enzyme is put together from preassembled sub- proteins, iron–sulfur clusters, and flavin mononucleotide cofactor complexes, and their subunit compositions have been characterized require the participation of assembly factors. Some are intrinsic to partially (14, 15). Extrinsic assembly factors of unknown function the complex, whereas others participate transiently. The suppres- become associated with subcomplexes that accumulate when as- sion of the expression of the NDUFA11 subunit of complex I dis- sembly and the activity of complex I are impaired by pathogenic rupted the assembly of the complex, and subcomplexes with mutations. Some assembly factor mutations also impair its activ- masses of 550 and 815 kDa accumulated. Eight of the known ex- ity (16). Other pathogenic mutations are found in all of the core trinsic assembly factors plus a hydrophobic protein, C3orf1, were subunits, and in 10 supernumerary subunits (NDUFA1, NDUFA2, associated with the subcomplexes. The characteristics of C3orf1, of NDUFA9, NDUFA10, NDUFA11, NDUFA12, NDUFB3, another assembly factor, TMEM126B, and of NDUFA11 suggest that NDUFB9, NDUFS4, and NDUFS6) (17–26). Those in supernu- they all participate in constructing the membrane arm of complex I. merary subunits NDUFA2, NDUFA10, NDUFS4, and NDUFS6 are associated with a reduced level of intact complex and accu- mitochondria | respiratory chain | NADH:ubiquinone oxidoreductase mulation of subcomplexes, indicating a defect in assembly or sta- bility of the complex, or both. n mammalian mitochondria, complex I (NADH:ubiquinone As described here, suppression of the expression of the su- Ioxidoreductase) provides the entry point for electrons from pernumerary membrane subunit NDUFA11 impairs assembly of NADH into the electron transport chain. For each two electrons complex I, leading to the accumulation of subcomplexes with transferred from NADH to ubiquinone, four protons are ejected estimated molecular masses of 550 and 815 kDa associated with from the mitochondrial matrix, thereby contributing to the pro- eight known assembly factors, plus three other proteins, espe- ton motive force across the inner membrane (1). The mamma- cially C3orf1. NDUFA11 has the characteristics of an intrinsic lian enzyme has 44 subunits, with a combined molecular mass of assembly factor for complex I and together with C3orf1 and about 1 MDa, assembled into an L-shaped complex, with one another extrinsic assembly factor TMEM126B (27), they prob- arm embedded in the inner membrane and the other protruding ably help to assemble the membrane arm of the complex. into the matrix of the organelle (2–4). Seven hydrophobic sub- units (ND1–ND6 and ND4L) of the membrane arm of NADH Results dehydrogenase (complex I) are encoded in mitochondrial DNA, Suppression of Expression of NDUFA11. The experiment, conducted and synthesized on mitochondrial ribosomes (5). The remainder in 143B cells, had three effects. First, cellular oxygen consump- are nuclear gene products, made in the cytoplasm and imported tion linked to complex I was reduced by two-thirds (Fig. 1), and into mitochondria (6). The seven proteins encoded in mito- chondrial DNA and seven nuclear-encoded subunits conserved Significance in prokaryotic complexes I, constitute the catalytic cores of the membrane and peripheral arms, respectively (1). The latter Mammalian complex I, the largest and most complicated en- contains binding sites for NADH, the primary electron acceptor zyme of the mitochondrial respiratory chain, is an L-shaped FMN, and seven iron–sulfur clusters that link FMN and the assembly of 44 proteins with one arm in the mitochondrial terminal electron acceptor, ubiquinone, bound at the juncture matrix and the orthogonal arm buried in the inner membrane. between the peripheral and membrane arms (1, 7). The mem- It is put together from preassembled modules. This inves- brane arm has four antiporter-like domains that probably provide tigation concerns the little studied process of the assembly of pathways for translocating protons (1). The remaining 30 so- the membrane arm module from proteins emanating from both called supernumerary subunits of mammalian complex I have no nuclear and mitochondrial genomes. We have identified two direct role in catalysis (2). Their functions are mostly unknown, membrane protein assembly factors C3orf1 and TMEM126B, not but they may be involved in assembly, stability, or regulation of found in the mature complex, that help this process by putting the complex (2, 8). together two membrane arm subcomplexes. Defects in the as- There is no atomic structure for any eukaryotic complex I, but sembly of complex I are increasingly being associated with human the arrangement and folds of the 14 core subunits in the mam- pathologies. malian enzyme are likely to be closely similar to those of bacterial orthologs, with the supernumerary subunits attached peripher- Author contributions: B.A. and J.E.W. designed research; B.A., J.C., S.D., and I.M.F. per- ally around the core (2, 9). The distribution of supernumerary formed research; B.A., J.C., S.D., I.M.F., and J.E.W. analyzed data; and J.E.W. wrote the subunits in bovine complex I is known from the subunit com- paper. positions of subcomplexes (3, 10, 11). Subcomplex Iλ is the pe- The authors declare no conflict of interest. ripheral arm, subcomplex Iα is a combination of Iλ and the Freely available online through the PNAS open access option. adjacent region of the membrane arm, subcomplex Iβ is the 1To whom correspondence should be addressed. E-mail: [email protected]. γ distal region of the membrane arm, and subcomplex I is an- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. other fragment from the membrane arm. The supernumerary 1073/pnas.1319247110/-/DCSupplemental. 18934–18939 | PNAS | November 19, 2013 | vol. 110 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1319247110 Downloaded by guest on September 28, 2021 A protein profile of complex I accompanying the eightfold reduction control of NDUFA11 correspond to additional proteins associated with P < 0.01 incompletely assembled complex I. Eight known assembly factors, NDUFA11-1 NDUFAF1–4, ACAD9, ECSIT, FOXRED1, and TMEM126B were associated with the NDUFA11-deficient subcomplexes (Fig. NDUFA11-2 3andDataset S1). FOXRED1 has been designated as an as- sembly factor from genetic and functional data (29), but here its 0 0.2 0.4 0.6 0.8 direct association with assembly intermediates of complex I has OCR (fmol/min/cell) been demonstrated. Three other proteins, C3orf1, ATP5SL, and B DNAJC11 were found. C3orf1 and ATP5SL have no ascribed control functions, whereas DNAJC11 belongs to the Hsp40 chaperone family. Both C3orf1 and TMEM126B colocalized with Mito- NDUFA11-1 Tracker, and so they are intrinsic mitochondrial proteins (Fig. S3). NDUFA11-2 Ablation of C3orf1, TMEM126B, ATP5SL, and DNAJC11 and Assembly of Complex I. The suppression of expression of C3orf1 or TMEM126B 3210 4 reduced both the cellular oxygen consumption, and the level of OCR (fmol/min/cell) intact complex I (Fig. 4), and subcomplexes of 315 and 370 kDa Fig. 1. Oxygen consumption of 143B cells with ablated NDUFA11. Oxygen accumulated. They were detected with antibodies against the pe- consumption rate (OCR) 96 h after transfection with two siRNAs (NDUFA11-1 ripheral arm and the membrane arm component NDUFB8, re- and -2, each 30 nM) against NDUFA11 (black and dark gray) compared with spectively. The accumulation of the 370-kDa subcomplex was control cells (light gray). (A) Complex-I–dependent OCR of 143B cells de- concomitant with a reduction in a 550-kDa subcomplex (Fig. 4), termined by subtraction of rotenone-inhibited OCR values from initial val- presumably the same subcomplex accompanying suppression of ues. (B) Addition of rotenone and duroquinol and OCR derived from expression of NDUFA11 (Fig. 2). Also, when TMEM126B was activities of complexes III and IV. Error bars are SD. suppressed, the levels of both complexes III and IV increased (Fig. 4). As the analyses were performed in the presence of n-dodecyl- β-D-maltoside, which dissociates respiratory complexes into in- the effect was bypassed by addition of the complex III substrate, dividual enzymes, the increase in complexes III and IV is not due duroquinol. Second, the mitochondrial network observed in to their release from supercomplexes. In their experiments on control cells became fragmented (Fig. S1). Third, the amount of TMEM126B, Heide et al. reported an increase in complex III, but intact complex I was reduced, and subcomplexes with molecular not of complex IV (27). masses of 815 and 550 kDa accumulated (Fig. 2). Previously The complex I assembly factors NDUFAF3, ACAD9, and known as the 830- and 650-kDa subcomplexes, respectively (16), NDUFAF2 are associated with subassemblies with masses of their sizes have been reestimated (Fig. S2). This accumulation of 400, 460, and 830 kDa, respectively (15). Based on the reesti- the 815-kDa complex is similar to the effects of mutation of mated masses of subcomplexes (Fig. S2) they probably corre- NDUFS4 or NDUFAF2 (28), but the accumulation of the 550- spond to the 315-, 370-, and 815-kDa subcomplexes, respectively, kDa complex has not been reported.